Determination of Bacteriocin Genes and Antibacterial Activity of Lactobacillus Strains Isolated from Fecal of Healthy Individuals.

Lactobacillus species play a major role in gastrointestinal (GI) tract function, intestinal microbiota balance, and the immune system activity by exerting a strong activity against many intestinal pathogens. The aim of this study was to isolate Lactobacillus species from fecal samples, investigate their antimicrobial properties, and characterize their bacteriocin encoding genes. 48 fecal samples were grown in MRS broth and then MRS agar. The colonies grown in MRS agar were selected and identiﬁed by PCR. 72 Lactobacillus species were obtained from 434 lactic acid bacteria (LAB) strains. Approximately 40% of all Lactobacillus isolates had antimicrobial activity against one or more microorganisms and 17.4% of them were active against all four indicator bacteria. The frequency of bacteriocin encoding genes were 5 (6.9%), 3 (4.1%) and 5 (6.9%) for Gassericin A, Plantaricin S and Laf operon, respectively. pH alteration had no effect on antibacterial activity, but in the alkaline range these activities were reduced and the strains showed the highest antibacterial activity after 48 h incubation. These data indicate that the majority of isolates were susceptible to GI tract or belonged to other bacterial forms such as viable but nonculturable (VBNC). The detection of bacteriocin encoding genes in about only 6% of all Lactobacillus isolates seems to be due to the existence of many other bacteriocin encoding genes in Lactobacillus species which were not tested. Further study of the bacteriocin gene clusters, types, subtypes and the probiotic effect of these strains will contribute to a better characterization of these isolates.

In recent years, bacteriocins have attracted significant attention because of their potential use as safe additives for food preservation (3) and other applications such as treatment of pathogen diseases, cancer therapy and human health enhancement (4).
Bacteriocins are a heterogeneous group of ribosomally-synthesized pore-forming antibacterial peptides. These antibacterial peptides can be produced by many labs such as Leuconostocetc, Streptococcus, Lactococcus, Pediococcus, and Lactobacillus that enable these bacteria to inhibit the growth and activity of several pathogenic bacteria (5).
Lactobacilli have many different bacteriocins with the same activity but vary in some aspects such as mode of action, genetic origin (chromosomal or plasmid) and biochemical properties.These peptides are being tested to assess their application as narrow-spectrum antibiotics and nowadays, play an effective role in processing and packaging food and have major potential as natural preservatives (6,7).
Based on killing mechanism, molecular weight, genetics and chemical properties, bacteriocins are classified in one of the four major groups: Class I (small peptide inhibitors termed as lantibiotics), Class II (small heat-stable proteins that have five subclasses), Class III (large, heatlabile protein and this class have two subclasses) and Class IV bacteriocins that are defined as complex bacteriocins containing lipid or carbohydrate sections (8).
Lactobacilli usually produce the highest volume of bacteriocins during the maximum cell growth, but some environmental factors such as pH, temperature, incubation period and media composition can influence on the volume of bacteriocin production (9).
In this study, Lactobacillus species were isolated from fecal samples and their antimicrobial activities against pathogen microorganisms were determined. Moreover the genes encoding bacteriocins were investigated while the antibacterial activity was partially characterized.

Materials and methods
Bacterial strains and growth conditions

Screening for antibacterial activity Agar spot test
The antibacterial activity of the isolates was determined by agar spot test according to Hernández et al. (11). Briefly, the strains were cultured in 5 ml of MRS broth at 37 ° C for 18 h and then, 3 μl aliquots of the culture were spotted onto agar plates containing 10 ml of MRS medium.  (11).

Identification of fecal isolates
434 LAB strains were isolated from volunteers with healthy fecal samples. Among these isolates, 72 (16.5%) were identified as Lactobacillus species based on 16S rRNA gene amplification.

Screening for antibacterial activity
All 72 Lactobacillus species were assayed for potential bacteriocin production and their antimicrobial activity by using the agar spot test and well-diffusion method. The inhibitory activity of lactobacilli strains against indicator bacteria is reported in Table 2.

Detection of the bacteriocin genes
All Lactobacillus species were tested for the presence of bacteriocin genes (Gassericin A, Plantaricin S and Laf operon). The frequency of these genes were 5 (6.9%), 3 (4.1%) and 5 (6.9%) for Gassericin A, Plantaricin S (structural gene) and Laf operon, respectively ( Figure 1). We did not identify strains having at least two of the three tested genes.
Partial characterization of the antibacterial activities pH alteration between 4 to 7 had no effect on antibacterial activities which arose due to the presence of bacteriocin components, but in the alkaline range (8 to 10), these activities were reduced. Regarding the effect of incubation time, the results showed that after 48 h incubation, the strains showed higher antibacterial activity in comparison with other incubation times tested.

Discussion
In Some environmental factors such as pH, temperature, incubation period and media composition can impact on the volume of bacteriocin production. Our results showed that pH alteration between 4 to 7 had no effect on antibacterial activity and these activities arose from bacteriocin components, but in the alkaline range (8 to 10), these activities were reduced. These results studies were performed at different pH and temperature on bacteriocin production and activity (17,18).
The study of the bacteriocin gene clusters, types, subtypes and the probiotic effect of these strains on laboratory animals will help to a better characterization of lactobacilli isolates.